Various analytical procedures and devices are commonly employed in flow-through assays to determine the presence and/or concentration of analytes that may be present in a test sample. For instance, immunoassays utilize mechanisms of the immune systems, wherein antibodies are produced in response to the presence of antigens that are pathogenic or foreign to the organisms. These antibodies and antigens, i.e., immunoreactants, are capable of binding with one another, thereby causing a highly specific reaction mechanism that may be used to determine the presence or concentration of that particular antigen in a biological sample.
There are several well-known immunoassay methods that use immunoreactants labeled with a detectable component so that the analyte may be detected analytically. For example, “sandwich-type” assays typically involve mixing the test sample with detectable probes, such as dyed latex or a radioisotope, which are conjugated with a specific binding member for the analyte. The conjugated probes form complexes with the analyte. These complexes then reach a zone of immobilized antibodies where binding occurs between the antibodies and the analyte to form ternary “sandwich complexes.” The sandwich complexes are localized at the zone for detection of the analyte. This technique may be used to obtain quantitative or semi-quantitative results. Some examples of such sandwich-type assays are described in. by U.S. Pat. No. 4,168,146 to Grubb, et al. and U.S. Pat. No. 4,366,241 to Tom, et al. An alternative technique is the “competitive-type” assay. In a “competitive-type” assay, the label is typically a labeled analyte or analyte-analogue that competes for binding of an antibody with any unlabeled analyte present in the sample. Competitive assays are typically used for detection of analytes such as haptens, each hapten being monovalent and capable of binding only one antibody molecule. Examples of competitive immunoassay devices are described in U.S. Pat. No. 4,235,601 to Deutsch, et al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler, et al.
Despite the benefits achieved from these devices, many conventional lateral flow assays encounter significant inaccuracies when exposed to relatively high analyte concentrations. For example, assays that rely on optical detection (e.g., fluorescence, reflectance, phosphorescence, etc.) often become inaccurate at high analyte concentrations. Specifically, the probes are usually not only captured on the surface of the membrane device, but also within the interior of the assay device. Unfortunately, most optical detection techniques are not capable of detecting those probes captured deep within the interior of the assay device. In addition, fluorescent probes sometimes exhibit “self-quenching” when placed too close together. Self-quenching is a well-known phenomenon that occurs when two or more fluorescent materials interact photochemically to quench each other's fluorescence. Thus, fluorescent probes may begin to exhibit self-quenching at high analyte concentrations, which actually results in a decrease in the fluorescent intensity. Those problems often limit the detection range and result in an inaccurate detection of an analyte.
In response to these or other problems, several assays configurations have been proposed. For example, EP 0462376 to Ching describes an assay device that includes a solid phase having at least two defined and marked detection sites in sequential fluid-flow contact. The first detection site is a capture site immobilized with a capture reagent capable of competing with the analyte for binding to a conjugate. A second detection site is a conjugate recovery site that includes a conjugate recovery agent different from the capture reagent for binding to the conjugate or a complex thereof that passes through the capture site. As the amount of analyte in the test sample increases, the more the bonding sites of the conjugate are occupied by analyte molecules and the less the conjugate is free to bind to the capture reagent. Instead, the analyte/conjugate complexes pass through the capture site and migrate into the conjugate recovery site. A comparative analysis of the amounts of label at each site indicates the amount of analyte in the test sample.
Nevertheless, a need still exists for a method of extending the dynamic detection range of an assay device in an accurate, yet simple and cost-effective manner.